Different microorganisms, among which bacteria and yeasts can be used as host organisms for different plasmids containing a DNA molecule comprising a nucleotide sequence coding for a specific protein. Among these microorganisms, yeasts are presently preferred and currently used. For example, European Patent application publication No. 0 106 828 discloses the use of Saccharomyces cerevisiae strains for the production of hepatitis B virus surface antigen (HBsAg) and European Patent application publication No. 0 103 409 relates to the production of alpha-1-antitrypsin from yeast plasmids.
Protein production in yeast strains indeed presents substantial advantages over production in bacterial strains. These advantages result from the rather easy growth of yeasts in large scale fermentors and from the fact that, contrary to bacteria, yeasts do appear to ressemble mammalian cells in their capacity to add carbohydrate groups to newly synthesized proteins.
Nevertheless the extraction and purification of the protein from a yeast culture do present technical problems due to the rather complex chemical composition of the yeast cell and more particularly to the presence of high lipid levels when the yeast growth is extended in order to improve the polypeptide production yield.
For example, the Saccharomyces cell wall is thought to consist of 3 layers: (a) an inner layer of alkali-insoluble .beta.-glucan, (b) a middle layer of alkali-soluble .beta.-glucan and (c) an outer layer of glycoprotein in which the carbohydrate consists of phosphorylated mannan; beneath the cell wall is a cytoplasmic membrane consisting of a very complex mixture of neutral lipids (mono-, di- and tri-glycerides), free and esterified sterols, a complex sphingolipid, glycerophosphatides and neutral as well as acidic glycolipids; the nucleus contains DNA, various species of RNA and a polyphosphate; vacuoles may contain a great variety of components of both high and low molecular weights, they serve as storage vesicles for a number of hydrolytic enzymes; the mitochondria are rich in lipid, phospholipid and ergosterol components of the membrane system and the cytoplasm contains a.o. large quantities of ribosomes, polyphosphates, glycogen and a number of glycolytic enzymes. Most yeast cells (such as Saccharomyces species) also contain some amount of lipid in the form of globules the amount of which does increase in extended cultures.
A number of multi-steps processes have been disclosed for the extraction and purification of proteins from different sources. Examples referring to proteins produced by engineered microorganisms are those published by Th. STAEHELIN et al. (J. Biol. Chem. 256; 9750-54; 198), K. MURRAY et al. (The EMBO J. 3; p. 645-650; 1984) and R. A. HITZEMAN et al. (Nucl. Ac. Res. 11; 2745-2763; 1983).
Typically, when the produced protein is cell bound, those different processes involve 3 series of steps.
In the first series of steps, the desired protein is removed from the cell interior. Therefore, the cells are either lysed (e.g. by enzymatic treatment) or disrupted (e.g. by mechanical forces (such as shearing forces (e.g. X-press or French press) or shaking with glass beads, eventually with addition of a detergent (see for instance K. MURRAY et al. loc. cit. and R. A. HITZEMAN et al. loc. cit.).
In the second series of steps, the medium is enriched in desired protein, e.g. by fractional precipitation by addition of ammonium sulfate and/or in the presence of polyethylene glycol.
Finally, in the third series of steps, substantially all contaminants are eliminated from the medium, e.g. by one or several operations selected from the group comprising ultrafiltration, ion exchange, gel filtration chromatography and isopycnic gradient centrifugation.
In such process, it is obvious that contaminants like accompanying proteins (cited by R. A. HITZEMANN et al. loc. cit.), nucleic acids and lipids and more particularly high lipid levels have a harmful influence on at least one of the steps of the third series (e.g. ultrafiltration and column chromatography) and the proteolytic enzymes must be eliminated rapidly from the medium. Moreover, it has also been noticed that ammonium sulfate precipitation is not possible without a previous rough delipidation because lipids interfere with this precipitation.
Therefore, it is of prime importance to dispose of a method wherein most of the contaminants are eliminated before the third series of steps.
Some among the previously described processes disclose the use of polyethylene glycol as a selective precipitating agent for proteins and a method for the precipitation of lipoproteins from plasma by using lipoprotein-polyanion-metal interactions has also been reported.
The method for fractional precipitation of proteins by using nonionic water-soluble polymers, in particular polyethylene glycol (PEG) has been introduced by POLSON et al. (Biochem. Biophys. Acta 82; 463-475; 1964) and discussed by different authors. Among them W. HONIG et al. (Analyt. Biochem. 72; 502-512; 1976) mention that "the specificity of precipitation, that is the ratio of desired protein and total protein, can be improved by using PEG fractions of lower average molecular weight than the generally employed PEG 6000". Nevertheless although "by manipulation of pH concentrates of individual plasma proteins may be obtained" the authors added "however, purification of more complex protein mixtures such as the supernatant of a cell homogenate is considerably poorer".
P. R. FOSTER et al. (Biochim. Biophys. Acta 317; 505-516; 1973 have described a method for the precipitation of enzymes from cell extracts of Saccharomyces cerevisiae by PEG. Methods for the concentration and purification of viruses and bacteriophages with PEG have been disclosed by B. P. VAJDA (Folia Microbiol. 23, 88-96; 1978) and G. J. LANCZ (Arch. Virusforsch. 42; 303-306; 1973). In the field of hepatitis antigen isolation, the purification of hepatitis B surface antigen (HBsAg) by a method comprising two successive treatments with PEG 6000 has been described by Ph. ADAMOWICZ et al. (p. 37-49 INSERM SYMPOSIUM No. 18, HEPATITIS B VACCINE, Publ. ELSEVIER, Amsterdam, Holland, 1981). In this method of HBsAg purification from plasma, immune complexes and most of the lipoproteins are, in a first step, precipitated from serum by PEG 6000 at a concentration of 5.5% and, in a second step, HBsAg is precipitated from the isolated supernatant by addition of PEG at a final concentration of 10%.
In the patent literature,
U.S. Pat. No. 3,790,552 discloses a method for removing hepatitis-associated antigen from a protein fraction which comprises a step wherein PEG having a molecular weight 200-6,000 is used in an amount of 12-30% (w/v) for precipitating said antigen. PA0 U.S. Pat. No. 3,951,937 discloses a process for the purification of hepatitis B antigen (HBAg) involving a double precipitation of HBAg with PEG (4.0-4.5 weight percent) having a molecular weight of at least 600. PA0 U.S. Pat. No. 3,994,870 discloses a method for purifying hepatitis B antigen (HBAg) wherein HBAg is precipitated by addition of 4.0-4.5 weight percent PEG and thereafter subjected to affinity chromatography utilizing insoluble concavalin A as a chromatographic adsorbent. PA0 European patent application, publication No. 0 112 506 discloses a process for producing a hepatitis B infection preventing vaccine from plasma comprising ammonium sulfate precipitation followed by adsorption on colloidal silicate and two successive precipitation steps with PEG (having a molecular weight of 2,000-10,000) at a 3-7% (w/v) to precipitate hepatitis B virus and immune complexes and at a 15-20% (w/v) in the supernatant to precipitate HBsAg. PA0 In the field of alpha-1-antitrypsin isolation, Japanese patent application No. 9128-335 (Derwent abstract 84-217127) discloses the precipitation of alpha-1-antitrypsin from plasma fraction by addition of PEG in an amount of 15-20% (w/v).
As mentioned above, a method for the precipitation of lipoproteins by using lipoprotein-polyanion-metal interactions has also been previously reported (for instance: M. BURSTEIN et al. Adv. Lip. Res. 11; 67-108; 1973 and A. VAN DALEN et al. Biochim. et Biophys. Acta 147; 421-427; 1967). This method is performed by interaction between the lipoproteins, a bivalent metal cation and an acidic polysaccharide and, in these operative conditions, the amount of precipitate is a function of the bivalent metal cation concentration in the medium.